A highly basic “KGRKPR” sequence in the coat protein of Cucumber necrosis virus plays an important role in the assembly of virions and encapsidation of full length viral RNA
Alam.S.B., Reade, R. and Rochon, D (2016) A highly basic “KGRKPR” sequence in the coat protein of Cucumber necrosis virus plays an important role in the assembly of virions and encapsidation of full length viral RNA. 35th Annual Meeting of the American Society for Virology, Virginia Tech, Blacksburg, Virginia
Plain language summary
Viruses must be able to successfully encapsulate the viral genome in order to produce stable virus particles that are capable of inducing virus infection. We have found that the virus coat protein which encapsulates the viral genome contains a series of positively charged amino acids that line the interior of the virus particle where the genetic material is found. These amino acids were postulated to play important roles in binding negatively charged viral genetic material. To test this hypothesis, these amino acids were mutated to uncharged residues and it was found that the virus was not capable of encapsulating complete viral genomes and moreover that the virus encapsulated the host genetic material rather than viral genetic material. In addition, the mutations produced particles which were not as stable as the non-mutated virus. Thus, we have identified amino acids in the virus that are important for proper virus particle assembly. This information can assist in the design of methods for management of virus disease.
CNV is a T=3 virus composed of 180 identical coat protein (CP) subunits and a single molecule of viral RNA (vRNA). The cryoEM structure of the CNV particle suggests that it consists of two concentric shells with vRNA sandwiched between the shells. Previous work in our lab has identified a highly basic “KGKKGK” region in the RNA binding domain of the CP that plays an important role in the encapsidation of full-length vRNA and in particle morphology. The epsilon region of the CNV CP arm contains a similar highly basic “KGRKPR” region which in C subunits lines the inner side of the outer shell at the 2-fold axis and coincides with vRNA. The present study investigates the importance of the epsilon region in vRNA binding and assembly. Multiple sequence alignment of this region across several genera in the Tombusviridae showed conservation of basic aa residues. Seven different mutants were constructed in the CNV epsilon region in which the basic residues were mutated to alanine individually, in pairs or in total. Genomic RNA and CP of all mutants accumulated to levels similar to that of WT CNV in infected plants, however particle assembly was compromised being more pronounced in mutants with two or four aa substitutions compared to single mutants. TEM of mutants showed that most contained a significantly higher level of T=1 and intermediate-sized particles when compared to CNV. Virion RNA analysis showed that significantly more truncated RNA species are encapsidated in double or quadruple mutants relative to WT or single mutants suggesting that encapsidation of full-length vRNA requires most of the basic aa residues in the epsilon region. Double mutants were also less thermally stable indicating protein-RNA interactions play an important role in virion stability. The nature and origin of truncated RNA species in mutants is being further investigated.